The invention relates to a substrate device which is configured for the cryopreservation of a biological sample containing biological cells, and which comprises in particular a cultivation surface for receiving the biological sample and a chamber for receiving the cultivation surface and a cultivation liquid. The invention further relates to a cryopreservation apparatus provided with at least one such substrate device. The invention further relates to a method for the cryopreservation of a biological sample containing biological cells which is carried out with said substrate device or the cryopreservation apparatus. The invention is used for the cryopreservation of biological cells, in particular for the cryopreservation of stem cells, e.g. human embryonic stem cells, or of germ cells, e.g. oocytes.
It is known that, for long-term storage, human embryonic stem cells (hESC) are subjected to cryopreservation (storage in the frozen state). One designated method, for example, is “slow rate freezing”, where tried-and-tested cryopreservation methods that work with other cell types are adapted for hESC. The cells are detached from a cultivation surface and frozen in suspension in sample vessels at slow cooling rates with the addition of cryoprotective agents (e.g. 10% DMSO). The samples are recovered by thawing e.g. in a waterbath (cf. e.g. B. C. Heng et al. in “Biotechnol. Appl. Biochem.” vol. 41, 2005, pp 97-104). The “slow rate freezing” method has disadvantages in terms of a low efficacy and reliability, chemical and mechanical stress when the cells detach from the cultivation surface, a low survival rate of the cells and a limited functionality of the thawed cells. Thus, after thawing, the cells can only grow to a limited extent on cultivation surfaces, requiring long recultivation times.
One known alternative to “slow rate freezing” is cryopreservation by vitrification (almost instant freezing or rapid freezing), where the cells are frozen at extremely fast cooling rates in order to achieve vitrification (at e.g. 130° C.). Cooling rates are assessed e.g. by M. Sansinena et al. in “Cryobiology” vol. 63(1), 2011, p. 32 (“Numerical simulation of cooling rates in vitrification systems used for oocyte cryopreservation”). A general disadvantage of vitrification is that, to avoid the formation of ice crystals, high concentrations of cryoprotective agents are required; however, these often have toxic effects and impair the result of the cryopreservation.
Because of the extremely fast cooling rates, conventional vitrification is restricted to small volumes of samples (cells and cryomedium). It has generally been the case hitherto that the smaller the volume, the larger is the surface-to-volume ratio of the sample, and the smaller the distance between the sample and a cooling medium, the greater is the probability of successful vitrification.
Another general limitation of conventional vitrification derives from the Leidenfrost phenomenon, which is distinguished by the formation of gas bubbles when warm surfaces come into contact with liquid nitrogen. Insulating regions can form which reduce the cooling rate and hence the chance of successful vitrification.
Furthermore, conventional vitrification makes high demands on the materials, which are expected to tolerate large and rapid temperature differences. In the case of vitrification substrates, the cracking or displacement of components can cause cell damage or reduce sample sterility. It is therefore of interest to find suitable materials or methods that prevent material damage and minimize wear.
One known method, for example, is in-straw vitrification, wherein the cells are detached from the cultivation surface and, after incubation in a cryomedium, transferred to an open or closed straw, optionally on the tip of a plastic pin (G. Vajta et al. in “Mol. Reprod. Dev.” vol. 51, 1998, pp 53-58; M. Richards et al. in “Stem Cells” vol. 22, 2004, pp 779-789; M. Kuwayama et al. in “Theriogenology” vol. 67, 2007, pp 73-80; and M. Kuwayama et al. in “Reprod. Biomed. Online” vol. 11, 2005, pp 608-614).
Although in-straw vitrification has good survival rates, it is unsuitable for large quantities. A very small sample volume and a maximized surface-to-volume ratio greatly reduce the quantity of cells that can be vitrified at one time. The thickness of the straw is very limited because the surface-to-volume ratio would become unfavorable for successful vitrification if the diameter were too large. Lengthening the straw would maintain the surface-to-volume ratio, but, because of the handling, would lead to very long sample incubation times and hence to cell damage.
Moreover, in-straw vitrification is very expensive and success is highly dependent on the individual expertise of the operator. Disadvantages of this method result in particular from the difficulty of handling the samples, which can give rise to inaccuracies in the adjustment of the incubation time in the highly concentrated, toxic cryoprotective agents and to high cell loss on freezing and thawing. Furthermore, the number of cells capable of being vitrified by these methods is very limited.
Another known vitrification method uses a so-called “cryoloop”. With the cryoloop, a droplet of sample containing the cells is held in a plastic ring at the end of a pin and immersed in liquid nitrogen (−196° C.) (M. Lane et al. in “Fertil. Steril.” vol. 72, 1999, pp 1073-1078). A disadvantage of this method results from the direct contact between the sample and the nitrogen and the danger of contamination of the cells by impurities in the nitrogen.
Another known vitrification method is adherent vitrification, wherein the cells in the adherent state are vitrified on a cultivation surface (A. F. Beier et al. in “Cryobiology” vol. 63, 2011, pp 175-185). The method proposed by A. F. Beier is illustrated schematically in FIG. 11 (state of the art). Firstly, biological cells 2′ are cultivated on a substrate platform 10′ in the adherent state, the substrate platform 10′ being arranged in a vessel 20′ containing a cultivation liquid 3′ (FIG. 11A). The cultivation liquid 3′ comprises e.g. a nutrient medium and at least one cryoprotective agent. For vitrification of the biological cells 2′, the substrate platform 10′ is transferred to another vessel 30′ containing liquid nitrogen as the cooling medium 4′ (FIG. 11B). Vitrification of the cells 2′ takes place in the vessel 30′. For permanent storage the substrate platform 10′ is placed in the vapor of the liquid nitrogen 4′ in a nitrogen tank 60′ (FIG. 11C).
This method again has a disadvantage resulting from the direct contact between the sample and the nitrogen and the consequent danger of contamination of the cells by impurities in the nitrogen. In particular, the possibility of clinical use is greatly restricted because of the risk of microbial contaminations (D. Stoop et al. in “Reprod. Biomed. Online” vol. 24, 2012, pp 180-185). Methods of sterilizing liquid nitrogen exist, but they are time-consuming and cost-intensive.
Although liquid nitrogen has a high purity directly after production and in practice is of pharmaceutical quality when sold commercially, contaminations due to microorganisms and other impurities can occur during transport and storage. Contaminations can even be transferred to the vapor phase due to aerosol formation on the surface of the liquid nitrogen, impairing the air quality. Methods of purifying liquid nitrogen by filtration have proved laborious and insufficiently reliable in practice.
There is an interest in vitrification methods that are easy to carry out, in particular with less stringent demands on precise observance of the incubation time in the cryomedium. There is also an interest in automating the cryopreservation so as to make the use of hESC more cost-effective, less labor-intensive and more efficient. Automated biobanks, for example, make it possible to store and use stem cells from a large number of patients or organisms.
For successful cryopreservation it is further desirable that, after vitrification, e.g. in a straw, and thawing, the cells be capable of growing again on a cultivation surface before they are made available e.g. for experiments or therapeutic use. There is an interest in minimizing the time between thawing and use so as to maximize the efficiency of the cryopreservation.
Many conventional cultivation methods do not allow the cells to be isolated or to be cultivated in the adherent state prior to cryopreservation. Hanging-droplet cultivation, which makes high demands on in situ cryopreservation, may be mentioned explicitly here. The possibility of novel cryopreservation techniques being usable with these cultivation techniques would bring great advantages and create scope for novel uses.
U.S. Pat. No. 5,257,128 discloses a cryobench for observing cells during freezing and thawing in a controllable liquid medium and at a controllable temperature ranging from 100° C. to −100° C. However, the cryobench is suitable neither for cultivation purposes nor for sample vitrification. DE 696 33 854 T2 discloses a method and a package for maintaining and storing cultivated tissue equivalents at low temperatures using a vessel consisting of a dish, a support with a membrane on which the tissue equivalent is immobilized, and a cover. Again this vessel is not suitable for cultivation purposes or sample vitrification. Cultivation vessels are described in U.S. Pat. No. 5,650,325, WO 9 640 858 A1 and GB 1 539 263 A, which, however are not designed for cryopreservation purposes.
An objective of the invention is to provide an improved substrate device and an improved method for the cryopreservation of a biological sample containing biological cells, said device and method eliminating or minimizing disadvantages and limitations of conventional techniques for the cryopreservation of biological samples. Another objective of the invention is to provide an improved cryopreservation apparatus provided with at least one substrate device for the cryopreservation of the biological sample. In particular, the invention should make available a cryopreservation technique by which a greater quantity of sample can be preserved at one time, which allows reproducible adjustment of the preservation conditions, which excludes potential sample contaminations and/or which allows vitrification of the biological samples.
These objectives are achieved by a substrate device, a cryopreservation apparatus and a method of the invention.
According to a first aspect of the invention, said objective is achieved by the general technical teaching of providing a substrate device, in particular for the cryopreservation of a biological sample containing biological cells, which comprises a substrate platform with a cultivation surface, and a first chamber which contains the cultivation surface of the substrate platform and is configured for receiving a cultivation liquid. According to the invention, the substrate device is provided with a second chamber which is configured for receiving a temperature control medium (cooling medium or heating medium). According to the invention, the first chamber and the second chamber are coupled together. Both chambers are connected to each other in an adjacent manner so that the substrate platform forms a separating wall between the interior of the first chamber and the interior of the second chamber. The first chamber (or first vessel, cultivation compartment) contains the cultivation surface having an areal, preferably flat extension. The cultivation surface is a surface which is made of a biologically compatible material suitable for receiving an adherent cell culture or a hanging-droplet culture. The second chamber (or second vessel, nitrogen compartment) is delimited from the first chamber by the substrate platform. The biological sample in the first chamber is isolated from the surroundings and in particular from the temperature control medium in the second chamber. An exchange between substances in the liquid or gaseous state is ruled out.
Advantageously, the substrate platform provided according to the invention between the first chamber and the second chamber fulfills several functions simultaneously. Firstly, the areal cultivation surface is provided on a front side of the substrate platform wherein the cultivation surface makes it possible to accommodate the biological sample with an extremely high surface-to-volume ratio. The size of the cultivation surface can be chosen without restrictions, so considerably greater quantities of sample can be subjected to cryopreservation than e.g. in the case of in-straw cryopreservation.
Secondly, the substrate platform is a solid component that extends along the laminar dimension of the cultivation surface. Perpendicular to the cultivation surface, i.e. in the direction of the thickness of the substrate platform, the latter extends an essentially smaller extend, thereby creating a negligible distance between the sample and the temperature control medium in terms of the transfer of heat from the temperature control medium to the biological sample. The substrate platform is in the form of a sheet, film or layer of material whose front side, facing towards the first chamber, provides the cultivation surface and whose opposite, back side, facing towards the second chamber, forms a closure with the second chamber.
Thirdly, the substrate platform ensures the separation of biological sample and temperature control medium, in particular the separation of biological sample and liquid nitrogen. Compared with conventional techniques, this affords novel uses of the substrate device with enhanced reliability, especially medical and biotechnological uses, without having to take special precautions to purify the temperature control medium.
According to a second aspect of the invention, the above-stated objective is achieved by the general technical teaching of providing a method for the cryopreservation of a biological sample wherein, in a first step, biological cells are arranged, in particular cultivated, on a cultivation surface of a substrate platform in a cultivation liquid in a first chamber, and in a second step, the temperature of the substrate platform is lowered and the biological sample is converted to a frozen state by filling a cooling medium into a second chamber, adjacent to the first chamber, the substrate platform forming a separating wall between the first chamber and the second chamber. Preferably, the method is carried out with the substrate device according to the above-mentioned first aspect of the invention. Preferably, by filling the cooling medium into the second chamber, the temperature of the substrate platform carrying the biological sample can be lowered rapidly in such a way as to achieve vitrification of the biological sample.
The method according to the invention can be carried out with different types of biological sample. The term “biological sample” denotes any composition of biological cells and a cultivation liquid. The cultivation liquid forms a liquid film or a liquid droplet around the cells. The biological cells include isolated cells, cell groups or cell colonies, in particular in the adherent or suspended state. The biological sample can comprise cells of one single type (identical cells) or cells of different types, e.g. stem cells and differentiated cells. In an advantageous variant of the invention, it is possible e.g. to subject cells of different types in the adherent state to a common cultivation (co-cultivation) on the cultivation surface before freezing takes place under the effect of the cooling medium. The cultivation liquid (cryomedium) generally comprises at least one nutrient medium and at least one cryoprotective agent, e.g. DMSO, propanediol or ethylene glycol. The cryoprotective agent can comprise in particular a composition of 20% DMSO, 20% ethylene glycol and 300 mM trehalose. It is possible to provide one single cultivation liquid at once or a succession of different cultivation liquids each containing different nutrient media and/or cryoprotective agents.
A particular advantage of the method according to the invention is that problems due to the Leidenfrost phenomenon, which were described above, are minimized with the technique according to the invention. Any gas bubbles eventually formed when the cooling medium is filled into the second chamber rise to the top of the second chamber, thus moving away from the substrate platform. The formation of unwanted regions of thermal insulation is therefore avoided.
According to a third aspect of the invention, the above-stated objective is achieved by the general technical teaching of providing a cryopreservation apparatus which comprises at least one substrate device according to the above-mentioned first aspect of the invention, and a rotating device which is configured for receiving and rotating (pivoting) the at least one substrate device. The substrate device is arranged in the cryopreservation apparatus in a rotatable manner. According to the invention, the substrate device can be rotated (pivoted) by means of the rotating device between different states that differ in terms of the arrangement of the first chamber and the second chamber in the vertical direction, i.e. in terms of the direction of gravity. By means of the rotating device the substrate device can be pivoted between a cultivation state, in which the first chamber is arranged above the second chamber in the vertical direction and the substrate platform forms the floor of the first chamber, and a temperature control state, in which the second chamber is arranged above the first chamber and the substrate platform forms the floor of the second chamber.
According to a preferred use of the invention, the substrate device is configured for vitrification of the biological sample on the cultivation surface. For this purpose the thickness and thermal conductivity of the substrate platform are preferably chosen so that, when the back side of the substrate platform, which is preferably exposed towards the interior of the second chamber, is wetted with a cooling medium at a temperature below the glass transition temperature of the sample, e.g. equal to or below −130° C., in particular with liquid nitrogen, the temperature of the biological sample is instantly brought to the temperature of the cooling medium. The glass transition temperature of the sample is e.g. around −130° C., but can be higher or lower depending on the concentration and the conditions, e.g. pressure. The thickness and thermal conductivity of the substrate platform are chosen in particular so as to achieve a cooling rate above minus 5000°/s, particularly preferably above minus 37,500°/s. Advantageously, this achieves cooling rates of practical interest (cf. above, M. Sansinena et al.).
Advantageously, the thickness of the substrate platform can be chosen by those skilled in the art, in particular according to the desired cooling rate, the lateral extent and the required mechanical stability. For the vitrification of the biological sample, it has proved advantageous if the substrate platform in a preferred variant of the invention has a thickness below 200 μm, particularly preferably below 120 μm, e.g. 100 μm or less. Moreover, the vitrification of the biological sample can advantageously be promoted if the substrate platform is made of glass, plastic, semiconductor material, e.g. silicon, or metal, e.g. copper, gold or silver. In general, biocompatible materials are used which have a high thermal conductivity, e.g. the thermal conductivities of the materials mentioned. A glass or plastic substrate platform has advantages in terms of a high mechanical stability and the availability of biocompatible materials. A substrate platform made of semiconductor material or metal also has advantages in terms of a high stability and in terms of a high thermal conductivity as well. Moreover, the use of metal for the substrate platform, or particularly preferably for the entire substrate device, can be advantageous by virtue of the thermal capacity of metals. Even if the cooling failed, e.g. in a nitrogen tank, the required preservation temperature of the sample could be maintained, at least temporarily, thereby minimizing sample losses.
According to another advantageous variant, the substrate platform can be made of a transparent material. Particularly preferably, the substrate platform can be formed in such a way that the biological sample on the cultivation surface can be subjected to an optical investigation, in particular a microscopic investigation. Advantageously, this allows the sample to be observed during freezing and during cryopreservation.
In another advantageous embodiment of the invention, the substrate device is provided with a substrate holder which is configured for a liquid-tight, detachable connection between the substrate platform and the first and/or second chamber. Advantageously, the substrate holder constitutes an anchoring means for exchangeable substrate platforms. The substrate device can be provided with different cultivation surfaces which can be chosen e.g. as a function of the cell types to be preserved and/or the use of the invention.
In another advantageous variant of the invention, the first and/or second chamber of the substrate device is provided with a compensating section. The compensating section is arranged between the substrate platform and the other parts of the first and/or second chamber and is configured for absorbing temperature-dependent mechanical stresses between the substrate platform and the first chamber. If the substrate platform and the other parts of the first or second chamber are made of different materials, the mechanical stresses which can arise when the substrate device is cooled or heated are compensated by the compensating section. The compensating section is e.g. an expansion joint, which forms a flexible buffer zone between the substrate platform and its holder in the first and/or second chamber.
Alternatively or additionally, the first chamber of the substrate device can be provided with a pressure equalizing valve. The pressure equalizing valve is adapted for equalizing any excess pressure between the first chamber and the surroundings. The excess pressure can occur e.g. when the substrate device is heated to recover the biological sample.
In another variant of the invention, the substrate platform can be an integral component of the first chamber or of the complete substrate device. For example, the substrate device can form a single piece with the first and second chambers and the substrate platform. In this case there are advantages in terms of the mechanical stability and compactness of the substrate device. The latter can be made of plastic by an injection molding process, for example.
The substrate device according to the invention advantageously allows different variants for the delivery of the cultivation liquid and/or a temperature control medium. For example, provision can be made for a manual delivery, wherein the respective media are filled into the first or second chamber with a pouring device. Alternatively, the substrate device can be provided with a delivery device which is configured for delivering the cultivation liquid and/or one of the temperature control media. This variant offers advantages for automated use of the substrate device and for increased reproducibility in the delivery of the media and in the observance of given preservation protocols. Advantageously, the delivery device can comprise e.g. a microfluidic device which is integrated into a wall or a cover of the first chamber or the second chamber. The microfluidic unit comprises e.g. a fluidic chip as known per se from microfluidic system technology, which has conducting and metering elements for media delivery. Alternatively or additionally, the delivery device can comprise at least one media line leading into the interior of the first chamber or second chamber. Like the microfluidic unit, the media line can alternatively be integrated into the substrate platform.
In another advantageous variant of the invention, if the first chamber and the second chamber of the substrate device are detachably connected to each other, there can be further advantages for the adaptation the substrate device to the requirement of a concrete use of the invention and for the handling of the substrate device, e.g. when cleaning and when loading the first chamber with the biological sample. In a first variant, the second chamber can be firmly connected to a chamber frame, which is configured for detachably receiving the first chamber. Advantageously, in this case, the second chamber with the chamber frame serves a dual function, firstly in terms of the temperature control of the biological sample in the first chamber, and secondly in terms of the holding of the first chamber. In another variant, the first chamber and the second chamber can be connected to each other via a screw joint.
The implementation of the invention is not restricted to the coupling of one single first chamber with one single second chamber. Particularly for the preservation of cells of different types, it can be advantageous if the first chamber is subdivided into several sub-chambers, each arranged for receiving a separate sample. The sub-chambers are arranged next to each other and adjacent to the second chamber, the substrate platform forming a common separating wall between the sub-chambers and the second chamber. Advantageously, all the samples in all the sub-chambers can be simultaneously brought to temperature, e.g. frozen or thawed, with the temperature control medium in the second chamber.
In another, particularly preferred embodiment of the invention, the substrate device is provided with a chamber holder, which is configured for receiving the substrate device in a pivotable manner in a support, in particular in the cryopreservation apparatus according to the third aspect of the invention. The chamber holder comprises e.g. two supporting elements arranged in a plane parallel to the extent of the substrate platform, which elements can be coupled with the support, e.g. the cryopreservation apparatus. The supporting elements are e.g. spigots which sit in bearings of the support, or pivot bearings for receiving spigots of the support. Advantageously, the chamber holder allows the substrate device to be pivoted rapidly and reproducibly about its lateral axis between the cultivation state and the preservation state.
Advantageously, the cryopreservation according to the invention can be carried out with different types of cell cultures, the cell cultures differing in terms of the provision of the biological cells on the cultivation surface. A first variant of the method according to the invention affords the cryopreservation of adherent biological cells, i.e. biological cells which are arranged on the cultivation surface in an adherent (sticking) manner. In this case, in a first partial step, the substrate device is placed in a cultivation state, in which the first chamber is arranged above the second chamber and the substrate platform forms the floor of the first chamber. The adherent cell culture on the cultivation surface is covered with the cultivation liquid. The biological cells are subjected on the cultivation surface to cultivation, i.e. to cell growth and optionally to cell proliferation under the action of nutrient media and/or differentiation factors in the cultivation liquid. In a further partial step, the substrate device is pivoted into a temperature control state, in which the second chamber is arranged above the first chamber and the substrate platform forms the floor of the second chamber. The cultivation liquid flows out of the first chamber so that, advantageously, the cells still adhering to the cultivation surface remain covered only with a thin liquid film formed due to the surface tension of the residual cultivation liquid. This minimizes the volume of the biological sample that is to be subjected to cryopreservation. In the temperature control state the cooling medium, e.g. liquid nitrogen, is filled into the second chamber. The cooling medium covers the upward-facing back side of the substrate platform so that the latter is instantly cooled together with the biological sample arranged on the cultivation surface.
A second variant of the method according to the invention affords a “hanging-droplet” cultivation and a vitrification of cells in the hanging droplet. This results in a cryopreservation of biological cells in a non-adherent state. The biological cells are frozen in hanging droplets. For this purpose, in a first partial step, the substrate device is placed in the temperature control state, in which the second chamber is above the first chamber and the substrate platform forms the floor of the second chamber. The cultivation surface of the substrate platform is aligned horizontally, the normal to the cultivation surface pointing vertically downwards, i.e. in the direction of gravity. Hanging droplets of the cultivation liquid are applied to the cultivation surface and biological cells are placed therein individually or in groups. Optionally, before freezing, provision can be made to cultivate the biological cells in the hanging droplets. In a second partial step, as in the first embodiment of the method according to the invention, the second chamber is filled with the cooling medium so that the substrate platform and the biological sample are rapidly frozen.
The substrate device according to the invention also offers advantages in the recovery of the cryopreserved cells. The second chamber can be used to thaw the biological sample. For this purpose a heating medium, for example water at a predetermined thawing temperature of e.g. 37° C., is filled into the second chamber when the substrate device is in the temperature control state. The substrate platform carrying the biological sample is heated by the heating medium until the biological sample is thawed. The cells can then be removed from the first chamber or subjected to further cultivation therein.
Further advantages of the invention are summarized below. The invention makes it possible to combine the advantages of adherent cryopreservation with those of vitrification by liquid nitrogen. As the cells can be cryopreserved in the adherent state, they do not need to be treated with enzymes, such as trypsin or collagenase, before vitrification, for instance to detach the cells from the substrate, and the colonies do not have to grow again, after thawing, before further culture is possible. Also, the cell-cell contacts are maintained in their original form, so the stress occurring on the cells is reduced even more.
Moreover, the survival rates and the functionality of the cells after cryopreservation are superior or comparable to those of in-straw vitrification. An advantage over the straw, however, is the possibility of preserving a large quantity of cells at one time. For example, a plurality of colonies can easily be vitrified in a simple manner by enlarging the cultivation surface of the substrate device. Also, the incubation times in which cryoprotective agents are introduced, optionally in high concentrations, are precisely definable since each cell colony comes into contact with the respective media simultaneously rather than at different times, as in the case of the straw, where each colony is treated individually.
Another advantage of the method according to the invention is that it can easily be automated. The samples do not have to be transferred from one cryomedium to the next by laborious manual pipetting and then sucked into the straw. Instead, the medium can be changed simply by suction or even by automatic rotation of the substrate. The ease of handling of the system ensures that the success of the preservation does not depend on the individual expertise and skill of the operator, but that the preservation can be carried out universally with similar success.
The danger of contamination of previous adherent vitrification methods is minimized by the “two-chamber system” according to the invention. Because there is at all times a physical barrier, in the form of the cultivation surface, between the cultivation compartment and the nitrogen compartment, no contact takes place between cell material and potentially non-sterile nitrogen. Moreover, the cultivation compartment (first chamber) can be separated from the surroundings, in particular protected from ambient air, by a cover. This simplifies the use of the system for therapeutic purposes and it is not necessary to use sterile nitrogen for vitrification or for storage in a tank.
The general physical conditions for successful vitrification, such as minimized sample volume and maximized surface-to-volume ratio for the fastest possible cooling rate, are favored by several aspects of the invention. Firstly, adherent cultivation and vitrification in the hanging state (“overhead”) results in the formation of a minimal liquid film over the cells. Excess medium flows out downwards and therefore does not lead to unwanted enlargement of the sample volume. Hence it is always possible to use sufficient cryomedium for incubation without the danger of too large a volume for vitrification. By virtue of the minimal liquid film, the toxic cryomedium can also be diluted easily with small amounts of a washing medium after thawing. Cell damage due to toxic cryomedia is thereby minimized. Moreover, the flat shape of the grown sample, e.g. cell colony, has a positive influence on the cooling rate. The distance between cooling medium and cells is minimal and the surface-to-volume ratio of the two-dimensional cell colony is greater than e.g. in the straw. In addition, the cell colonies can be cryopreserved in co-culture with other cell types. This dispenses with a labor-intensive preparation of the co-culture after thawing, and the time spent by the cells outside the co-culture is minimized. This advantage is particularly pronounced in the co-culture between hESC and mouse feeder cells.
The possibility of using different cultivation surfaces enables the substrate device according to the invention to be manipulated as a function of use and cell type. The success of vitrification can be further maximized by improved thermal conduction of the surface. A special anchoring means for different culture surfaces enables them to be exchanged as a function of use. Thermally induced volume changes in the materials used can be absorbed by a flexible buffer zone between the culture surface and its holder. This allows materials with different coefficients of thermal expansion to be used for the substrate. A possible alternative is to use the same material for the culture surface and the rest of the substrate device. The volume changes are therefore the same and no stresses occur in the material.
As the formation of a meniscus increases the volume of media over the cells in the marginal region of the substrate, the angle and the material in the marginal region can be adapted so that a meniscus is no longer formed. This favors optimization of the vitrification of the cells even in the marginal region.